Resistance of human pathogens to anti-infective agents poses a serious threat to human health and requires sustained efforts to develop new therapies. The essential methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is widespread in human pathogens, including some of the most deadly infections caused by M. tuberculosis and P. falciparum. Several of the enzymes in this pathway catalyze unprecedented reactions, making them particularly attractive as targets for the development of selective inhibitors. Our long term goal is to understand catalysis in the MEP pathway toward the development of inhibitors targeting isoprenoid biosynthesis in pathogens. This proposal describes studies to examine three intriguing MEP pathway enzymes, IspG, DXP synthase and IspF. Specific Aims 1 and 3 focus on mechanistic studies and inhibitor development of the enzymes that generate and utilize cyclodiphosphate intermediate, methylerythritol cyclodiphosphate (MEcPP). The goal of Specific Aim 2 is to understand catalysis of DXP synthase in C-N bond formation to generate the medicinally useful hydroxamic acid compound class. Anti-infective agents developed to target these enzymes have the potential to broadly impact the treatment of deadly infectious diseases.